This invention relates to a one pot synthetic method for the synthesis of 5xe2x80x2-hydroxy phosphorylated nucleoside derivatives.
The spread of AIDS and the ongoing efforts to control the responsible virus are well-documented. One way to control HIV is to inhibit its reverse transcriptase activity (RT). Thus, novel, potent, and selective inhibitors of HIV RT are needed as useful therapeutic agents. Known, potent inhibitors of HIV RT include 5xe2x80x2-triphosphates of 2xe2x80x2,3xe2x80x2-dideoxynucleoside (xe2x80x9cddNxe2x80x9d) analogues. Various 5xe2x80x2-hydroxy phosphorylated 2xe2x80x2,3xe2x80x2-dideoxynucleoside derivatives such as phenyl phosphate derivatives of 3xe2x80x2-azidothymidine (AZT), 2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxythymidine (d4T) and 3xe2x80x2-deoxythymidine (3dT) have long been tested as anti-HIV agents. Given the significance of ddN derivatives, development of new methodologies for their synthesis, especially syntheses amenable to large scale commercial production and affording a high yields, is important.
Despite intensive research regarding the preparation of ddN derivatives, the synthesis of 5xe2x80x2-hydroxy phosphorylated ddN derivatives has involved synthetic methodology requiring at least three separate synthetic steps each usually involving purification of the resulting intermediate. The most commonly used synthetic procedure affording 5xe2x80x2-hydroxy phosphorylated ddN derivatives is the three-step synthetic sequence shown in Scheme 1. 
The current methods for the synthesis of 5xe2x80x2-hydroxy phosphorylated ddN derivatives suffer from a variety of disadvantages. For example, the commonly used synthetic method of Scheme 1 involves multiple solvents and a variety of work up and purification procedures. Specifically, the method of Scheme 1 uses three different solvents for the three separate steps, namely Et2O in the first step, CHCl3 in the second step and THF in the third step. Additionally, the method of Scheme 1 requires purification of the p-bromophenyl phosphorodichloridate intermediate via distillati In at the end of step 1 and removal of HCl amine salts formed at the end of step 2. The use of these additional work up, and purification steps, as well as multiple reagents increases the production costs and decreases the yields of 5xe2x80x2-hydroxy phosphorylated ddN derivatives. Furthermore, existing synthetic methods for production of 5xe2x80x2-hydroxy phosphorylated ddN derivatives are not amenable to large scale commercial production.
Generally, the present invention relates to a method for preparing a 5xe2x80x2-hydroxy phosphorylated nucleoside compound in a single reaction vessel. The method provides a high yielding synthesis of 5xe2x80x2-hydroxy phosphorylated nucleoside compounds and related derivatives without the need for purification and isolation of intermediates, and is amenable to large scale synthesis.
The invention provides a method for preparing a 5xe2x80x2-hydroxy phosphorylated nucleoside compound in a single reaction vessel. Phosphorous oxychloride is contacted with a halophenol moiety to produce a halophenyl phosphorodichloridate. Without purification, the halophenyl phosphorodichloridate is reacted with a carboxy-protected amino acid to produce a halophenyl carboxy-protected amino acid phosphorochloridate. Without purification, the halophenyl carboxy-protected amino acid phosphorochloridate is reacted with a nucleoside.
One embodiment of the invention provides a method of preparing AZT-5xe2x80x2-(para-bromophenyl methoxyalaninyl phosphate) in a single reaction vessel without purification of the intermnediates formed. Phosphorous oxychloride is reacted with a para-bromophenol moiety to form para-bromophenyl phosphorodichloridate. Without purification, the para-bromophenyl phosphorodichloridate is contacted with alanine methyl ester to produce para-bromophenyl methoxyalaninyl phosphorochloridate. Without purification, the para-bromophenyl methoxyalaninyl phosphorochloridate is reacted with AZT.
Another embodiment of the invention provides a method of preparing d4T-5xe2x80x2-(para-bromophenyl methoxyalaninyl phosphate) in a single reaction vessel without purification of the intermediates formed. Phosphorous oxychloride is reacted with a para-bromophenol moiety to form para-bromophenyl phosphorodichloridate. Without purification, the para-bromophenyl phosphorodichloridate is contacted with alanine methyl ester to produce para-bromophenyl methoxyalaninyl phosphorochloridate. Without purification, the para-bromophenyl methoxyalaninyl phosphorochloridate is reacted with d4T.
The present invention is applicable to the preparation of 5xe2x80x2-hydroxy phosphorylated nucleoside derivatives. In particular, the present invention is directed to a method for the synthesis of 5xe2x80x2-hydroxy phosphorylated nucleoside derivatives in a single reaction vessel without the need for purification or isolation of reaction intermediates. While the present invention is not so limited, an appreciation of various aspects of the invention will be gained through a discussion of the examples provided below.
The method involves the synthesis of 5xe2x80x2-hydroxy phosphorylated nucleoside derivatives in a single reaction vessel by the sequential addition of various reagents. Synthesis of 5xe2x80x2-hydroxy phosphorylated 2xe2x80x2,3xe2x80x2-dideoxynucleoside derivatives is carried out in a single reaction vessel. Phosphorous oxychloride is contacted with a halophenol moiety to produce a halophenylphosphorodichloridate. Without purification, the halophenyl phosphorochloridate is reacted with a carboxy-protected amino acid to produce a halophenyl carboxy-protected amino acid phosphorochloridate. Without purification, the halophenyl carboxy-protected amino acid phosphorochloridate is reacted with a nucleoside.
First, a halophenyl phosphorodichloridate species is formed by contacting phosphorous oxychloride with a halophenol moiety in the presence of an organic solvent and a tertiary amine. Suitable halophenol moieties include a phenyl group substituted with 1 to 5 halogens selected from fluorine, chlorine, bromine, iodine, or a mixture thereof. Preferred halophenol moieties include para-halogenated phenol moieties. A most preferred halophenol moiety is a para-bromo phenol moiety.
The phosphorous oxychloride and halophenol moiety are typically contacted in an organic solvent which is suitable for every chemical transformation involved in synthesizing 5xe2x80x2-hydroxy phosphorylated 2xe2x80x2,3xe2x80x2-dideoxynucleoside derivatives in a single reaction vessel. Examples of suitable organic solvents include ethers, such as diethyl ether (Et2O), chlorinated solvents, such as methylene chloride (CH2Cl2), chloroform, or dichloroethane, and aromatics, such as toluene, or tetrahydrofurane (THF). Preferably, the organic solvent is THF or methylene chloride. The phosphorous oxychloride and halophenol moiety are typically contacted in the presence of a tertiary amine. Examples of tertiary amines include triethylamine (Et3N), 1-methylimidazole, trimethylamine, tri-n-propylamine, N,N-dimethylaniline, and triphenylamine. Preferably, the tertiary amine is triethylamine or 1-methylimidazole. Most preferably the tertiary amine is triethylamine.
The present method typically employs a molar ratio of POCl3 to the halophenol moiety of about 0.5:1 to about 1:3. Preferably, the molar ratio of POCl3 to the halophenol moiety is equal or a slight molar excess of the halophenol moiety is employed. Preferably, the molar ratio of POCl3 to the halophenol moiety is from about 1:1 to about 1:2. Most preferably, a POCl3 to halophenol moiety ratio of about 1:1 to about 1:1.2 is employed. Additionally, the molar ratio of tertiary amine to halophenol moiety is from about 0.5:1 to about 3:1. Preferably, the molar ratio of tertiary amine to halophenol moiety is from about 1:1 to about 2:1. Most preferably, the molar ratio of tertiary amine to halophenol moiety is from about 1:1 to about 1.2:1.
The reaction time necessary to form the halophenyl phosphorodichloridate species will depend on temperature, stoichiometric ratios of the reagents, and the halophenol reagent utilized. Generally, reaction times range from about 1 to 20 hours. Typically, reaction times range from about 12 to about 15 hours.
In the presence of a tertiary amine, the phosphorous oxychloride (POCl3) and halophenol moiety are initially contacted at about 0xc2x0 C. The resulting reaction mixture is preferably maintained at a temperature of about 25xc2x0 C. Temperatures of about xe2x88x9220xc2x0 C. to about 30xc2x0 C. are employed in order to maintain a convenient reaction rate without substantially decreasing yield.
A halophenyl carboxy-protected amino acid phosphorochloridate moiety is then formed by contacting the halophenyl phosphorodichloridate formed above with a carboxy-protected amino acid in the same reaction vessel. The carboxy-protected amino acid and the halophenyl phosphorodichloridate formed above are contacted in the presence of an organic solvent and a tertiary amine as defined above.
Carboxy-protected amino acids can be prepared from a natural or synthetic amino acid and a carboxy-protecting group using methods known to those skilled in the art. Typical methods for preparing carboxy-protected amino acids include, for example, the preparation of the methyl ester, ethyl ester, benzyl ester, methoxymethyl ester, and benzyloxymethyl ester. Suitable carboxy protective groups and methods for the preparation of carboxy-protected amino acids are described in Protective Groups in Organic Synthesis, Greene, ed., John Wiley and Sons, New York (1981) and The Peptides: Analysis, Synthesis, Biology: Vol.3: Protections of Functional Groups in Peptide Synthesis, E. Gross and J. Meinenhofer, eds., Academic Press, New York (1981), the disclosures of which are incorporated herein by reference.
Suitable carboxy-protected amino acids include carboxy-protected natural and synthetic amino acids. xe2x80x9cAmino acidxe2x80x9d refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers), synthetic analogs and derivatives thereof. xcex1-Amino acids include a carbon atom to which is bonded an amino group, a carbonyl group, a hydrogen atom, and a distinctive side chain. The side chains of naturally occurring amino acids are well known in the art and include, for exampie, hydrogen (e.g., as in glycine), alkyl (e.g. as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroalkyl (e.g., as in histidine). One of skill in the art will appreciate that the term amino acid also includes xcex2-, xcex3-, xcex4-, and xcfx89-amino acids, and the like. Synthetic amino acids such as, for example, alanine, glycine, valine, phenylalanine, cysteine, serine, lysine, glutamate, glutamine, trifluoroleucine, p-fluorophenylalanine, and 3-triethylalanine are also known in the art. Typically, the carboxy protected amino acids are esters of natural and synthetic amino acids. Preferably, the carboxy protected amino acid is an alanine ester. Most preferably the carboxy protected amino acid is alanine methyl ester.
The present method typically employs a molar ratio of carboxy-protected amino acid to POCl3 of about 0.5:1 to about 3:1. Preferably, the molar ratio of carboxy-protected amino acid to POCl3 is equal or a slight molar excess of the carboxy-protected amino acid is employed. Preferably, the molar ratio of carboxy-protected amino acid to POCl3 is from about 1:1 to about 2:1. Most preferably, a carboxy-protected amino acid to POCl3 molar ratio of about 1:1 to about 1.2:1 is employed. An additional amount of triethylamine is added to the reaction vessel subsequent to the addition of the carboxy-protected amino acid. This second portion of triethylamine is added at a molar ratio from about 0.5:1 to about 4:1, triethylamine to carboxy-protected amino acid. Preferably, the molar ratio of tertiary amine to carboxy-protected amino acid is from about 1:1 to about 2:1. Most preferably, the molar ratio of tertiary amine to carboxy-protected amino acid is from about 1:1 to about 2:1.
The reaction time necessary to form the halophenyl carboxy-protected amino acid phosphorochloridate species will depend on temperature, stoichiometric ratios of the reagents, and the halophenyl phosphorodichloridate reagent utilized. Generally, reaction times range from about 1 to 20 hours. Typically, reaction times range from about 12 to about 15 hours.
When forming the halophenyl carboxy-protected amino acid phosphorochloridate species temperatures of about xe2x88x9270xc2x0 C. to about 30xc2x0 C. are preferably employed in order to maintain a convenient reaction rate without substantially decreasing yield.
Finally, the desired 5xe2x80x2-hydroxy phosphorylated 2xe2x80x2,3xe2x80x2-dideoxynucleoside derivative is formed by contacting a halophenyl carboxy-protected amino acid phosphorochloridate formed above with the desired 2xe2x80x2,3xe2x80x2-dideoxynucleoside in the same reaction vessel. The halophenyl carboxy-protected amino acid phosphorochloridate and the desired 2xe2x80x2,3xe2x80x2-dideoxynucleoside are contacted in the presence of an organic solvent and a tertiary amine as defined above.
The present method typically employs a molar ratio of 2xe2x80x2,3xe2x80x2-dideoxynucleoside to POCl3 of about 0.25:1 to about 2:1. Preferably, the molar ratio of 2xe2x80x2,3xe2x80x2-dideoxynucleoside to POCl3 is from about 0.33:1 to about 1:1.2. Most preferably, the molar ratio of 2xe2x80x2,3xe2x80x2-dideoxynucleoside to POCl3 is from about 1:1 to about 1:1.2. An additional amount of triethylamine is added to the reaction vessel either subsequent to or concurrent with the addition of the 2xe2x80x2,3xe2x80x2-dideoxynucleoside. This third portion of triethylamine is added at a molar ratio from about 0.25:1 to about 7:1 triethylamine to POCl3. Preferably, the molar ratio of tertiary amine to POCl3 is from about 0.33:1 to about 6:1. Most preferably, the molar ratio of tertiary amine to POCl3 is from about 1:1 to about 6:1.
The reaction time necessary to form desired 5xe2x80x2-hydroxy phosphorylaed 2xe2x80x2,3xe2x80x2-dideoxynucleoside derivative will depend on temperature, stoichiometric ratios of the reagents, and the halophenyl carboxy-protected amino acid phosphorochloridate and 2xe2x80x2,3xe2x80x2-dideoxynucleoside reagents utilized. Generally, reaction times range from about 10 to 50 hours. Typically, reaction times range from about 24 to about 48 hours.
The halophenyl carboxy-protected amino acid phosphorochloridate and 2xe2x80x2,3xe2x80x2-dideoxynucleoside are initially contacted at about 20xc2x0 C. to 30xc2x0 C. The resulting reaction mixture is maintained at a temperature of about 20xc2x0 C. to 30xc2x0 C. Temperatures of about 0xc2x0 C. to about 30xc2x0 C. are typically employed in order to maintain a convenient reaction rate without substantially decreasing yield.
As used herein the term xe2x80x9chalophenol moiety,xe2x80x9d unless stated otherwise, refers to a phenol group substituted with 1 to 5 halogens.
As used herein the term xe2x80x9chalogen,xe2x80x9d unless stated otherwise, refers to fluorine, chlorine, bromine, or iodine.
As used herein the term xe2x80x9camino acidxe2x80x9d unless stated otherwise, refers to xcex1 and xcex2 amino derivatives of aliphatic carboxcyclic acids. The term xe2x80x9camino acidsxe2x80x9d includes both natural and synthetic amino acids such as, for example, alanine, glycine, valine, phenylalanine, cysteine, serine, lysine, glutamate, glutamine, trifluoroleucine, p-fluorophenylalanine, and 3-triethylalanine.
As used herein the term xe2x80x9cnucleoside,xe2x80x9d unless stated otherwise, refers to any glycoside that is a component of a nucleic acid and that consists of a nitrogenous base linked to a ribofuranose such as, for example, 2xe2x80x2,3xe2x80x2-dideoxynucleosides. Typically, nucleosides include those substituted at the 1xe2x80x2 position. Preferably, the substituent at the 1xe2x80x2 position is purine or pyrimidine. Most preferably the substituent at the 1xe2x80x2 position is thymine. Suitable nucleosides include those having the following formulae: 
One embodiment of the invention provides a method of preparing AZT-5xe2x80x2-(para-bromophenyl methoxyalaninyl phosphate) as shown in Scheme 2. The AZT-5xe2x80x2-(para-bromophenyl methoxyalaninyl phosphate) is prepared in a single reaction vessel without purification of the intermediates. 
Phosphorous oxychloride is reacted with a para-bromophenol moiety to form para-bromophenyl phosphorodichloridate. Without purification, the para-bromophenyl phosphorodichloridate is contacted with alanine methyl ester to produce para-bromophenyl methoxyalaninyl phosphorochloridate. Without purification, the para-bromophenyl methoxyalaninyl phosphorochloridate is reacted with AZT.
Another embodiment of the invention provides a method of preparing d4T-5xe2x80x2-(para-bromophenyl methoxyalaninyl phosphate) in a single reaction vessel without purification of the intermediates formed. Phosphorous oxychloride is reacted with a para-bromophenol moiety to form para-bromophenyl phosphorodichloridate. Without purification, the para-bromophenyl phosphorodichloridate is contacted with alanine methyl ester to produce para-bromophenyl methoxyalaninyl phosphorochloridate. Without purification, the para-bromophenyl methoxyalaninyl phosphorodichloridate is reacted with d4T.
The products formed using the methods of the invention can be used to form other chemical compounds. In one embodiment, the 5xe2x80x2-hydroxy phosphorylated nucleoside can be treated with a solution of bromine in methanol to produce, for example, a 5-bromo-6-methoxy-5,6-dihydro-AZT-5xe2x80x2-(halophenyl carboxy-protected amino acid phosphate) or 5-bromo-6-methoxy-5,6-dihydro-d4T-5xe2x80x2-(halophenyl carboxy-protected amino acid phosphate). Such molecules have demonstrated combined spermicidal and anti-HIV activity. For example, 5-bromo-6-methoxy-5,6-dihydro-AZT-5xe2x80x2-(p-bromophenyl methoxyalaninyl phosphate) has demonstrated an EC50 value of 2.8 xcexcM in sperm motility assays and an IC50 value of 0.005 xcexcM in HIV replication assays.